Our previous study demonstrated successful treatment of an established rat brain tumor through the bystander effect by intra-tumoral injection of neural stem cells transduced with herpes simplex virus-thymidine kinase gene (NSCtk) followed by systemic ganciclovir (GCV) administration (NSCtk therapy). Since glioma has a strong tendency to infiltrate into surrounding brain tissue and that is one of the main causes of local treatment failure, we, in the present study, injected NSCtk cells at distant sites of rat brain tumors and evaluated migratory potential of NSCtk toward the tumor and anti-tumor effects of the NSCtk therapy of this experimental setting. NSCtk cells were intracranially implanted either at 2mm medial in the ipsilateral hemisphere or at the mirror point in the contralateral hemisphere to the C6 rat glioma cell implantation. Active migration of NSCtk cells toward C6 cells was observed even when NSCtk cells were implanted in the contralateral hemisphere. When GCV was systemically administered, growth of intracranial tumor was markedly inhibited and the survival was significantly prolonged through the bystander effect by NSCtk cells migrated from distant injection sites of the tumor. The results of the present study suggest that NSCtk therapy is still effective in the area far from the NSCtk injection site and, therefore, suitable for treatment of malignant gliomas that deeply infiltrate and widely disseminate in the brain.